Nadh Assay Archives - Gen9 Genetics

An efficient in vivo regeneration of the primary cellular resources NADH and ATP is vital to optimize the production of value-added chemicals and enable the activity of synthetic pathways. Currently, these regeneration pathways are mainly tested and characterized in vitro before entering the cell. However, in vitro measurements can be misleading as they do not reflect enzyme activity under physiological conditions. Here, we build an in vivo platform to test and compare NADH regeneration systems. By removing dihydrolipoyl dehydrogenase in Escherichia coli, we remove the activity of pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. When grown in acetate, the resulting strain is auxotrophic for NADH and ATP: acetate can be assimilated through glyoxylate derivatization, but cannot be oxidized to provide the cell with reducing power and energy. This strain can therefore be used to select and test different NADH regeneration routes. We exemplify this by comparing various NAD-dependent formate dehydrogenases and methanol dehydrogenases. We identify the most efficient enzyme variants under in vivo conditions and determine the optimal raw material concentrations that maximize NADH biosynthesis and avoid cellular toxicity. Therefore, our strain provides a useful platform to compare and optimize enzyme systems for the regeneration of cofactors under physiological conditions.


The growth of an organism requires three general components: reducing power, energy and chemical elements, such as carbon and nitrogen. The first two are typically connected, since the transfer of reducing power from an electron donor to an acceptor (eg, oxygen, nitrate, or an organic compound) is coupled with energy conservation in the form of ATP biosynthesis. In heterotrophic metabolism, all types of components are derived from a single growth substrate, where its catabolism not only restores reducing power and energy, but also provides carbon components for cell growth. However, in some cases, the carbon source is decoupled from the power and energy reduction source. The most notable example is autotrophic growth, where two different systems are responsible for carbon fixation and regeneration of NAD (P) H and ATP. Another example is the use of auxiliary substrates that are not converted to biomass but are completely oxidized to provide the cell with more reducing power and energy, allowing a larger fraction of the primary substrate to provide carbon units for biosynthesis (Babel, 2009). This approach is also commonly used in cell-free systems, where the bioconversion process is compatible with parallel systems for the regeneration of NAD (P) H and ATP (Claassens, Burgener, Vogeli, Erb, and Bar-Even, 2019).

To optimize the in vitro regeneration of NAD (P) H and ATP, it is usually sufficient to choose an enzyme system with a high Vmax, which can be measured and compared under the specific relevant conditions. However, optimization of the in vivo regeneration of NAD (P) H and ATP is more delicate. First, the kinetic parameters of the enzyme can change substantially within the cellular environment, which is difficult to mimic in in vitro measurements (van Eunen and Bakker, 2014; van Eunen, Kiewiet, Westerhoff, and Bakker, 2012). In addition, the steady-state physiological concentrations of the substrates and products affect the thermodynamics and kinetics of the reaction (Noor et al., 2016). Finally, overexpressed enzymes, both native and heterologous, tend to bend and degrade, thus reducing, sometimes dramatically, the amount of functional protein in the cell (Roodveldt, Aharoni, and Tawfik, 2005). Therefore, there is a growing need to establish an in vivo platform to test and compare cofactor regeneration pathways.